Merck

Andrographolide sodium bisulphite-induced inactivation of urease: inhibitory potency, kinetics and mechanism.

BMC complementary and alternative medicine (2015-07-17)
Zhi-Zhun Mo, Xiu-Fen Wang, Xie Zhang, Ji-Yan Su, Hai-Ming Chen, Yu-Hong Liu, Zhen-Biao Zhang, Jian-Hui Xie, Zi-Ren Su
RESUMEN

The inhibitory effect of andrographolide sodium bisulphite (ASB) on jack bean urease (JBU) and Helicobacter pylori urease (HPU) was performed to elucidate the inhibitory potency, kinetics and mechanism of inhibition in 20 mM phosphate buffer, pH 7.0, 2 mM EDTA, 25 °C. The ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of ASB was characterized with IC50 values. Lineweaver-Burk and Dixon plots for JBU inhibition of ASB was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode. The IC50 of ASB against JBU and HPU was 3.28±0.13 mM and 3.17±0.34 mM, respectively. The inhibition proved to be competitive and concentration- dependent in a slow-binding progress. The rapid formation of initial ASB-JBU complex with an inhibition constant of Ki=2.86×10(-3) mM was followed by a slow isomerization into the final complex with an overall inhibition constant of Ki*=1.33×10(-4) mM. The protective experiment proved that the urease active site is involved in the binding of ASB. Thiol reagents (L-cysteine and dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while boric acid and fluoride show weaker protection, indicating that the active-site sulfhydryl group of JBU was potentially involved in the blocking process. Moreover, inhibition of ASB proved to be reversible since ASB-inactivated JBU could be reactivated by dithiothreitol application. Molecular docking assay suggested that ASB made contacts with the important sulfhydryl group Cys-592 residue and restricted the mobility of the active-site flap. ASB was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for the treatment of urease-related diseases.

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Supelco
Urea, 8 M (after reconstitution with 16 mL high purity water)
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L-Cysteine, produced by Wacker Chemie AG, Burghausen, Germany, ≥98.0%
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Boric acid, suitable for electrophoresis, ≥99.5%
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Boric acid, BioXtra, ≥99.5%
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Boric acid, tablet, 1 g boric acid per tablet
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Urea, suitable for electrophoresis
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Boric acid, 99.999% trace metals basis
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Urea, puriss., meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%, 99.0-101.0% (calc. on dry substance)
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Boric acid, 99.97% trace metals basis
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Sodium fluoride, 99.99% trace metals basis
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L-Cysteine, 97%
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L-Cysteine, ≥97%, FG
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Sodium fluoride, BioReagent, suitable for insect cell culture, ≥99%
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Sodium fluoride, BioXtra, ≥99%
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Urea, meets USP testing specifications
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Urea, ACS reagent, 99.0-100.5%
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Urea, ReagentPlus®, ≥99.5%, pellets
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Urea, BioXtra, pH 7.5-9.5 (20 °C, 5 M in H2O)
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Boric acid, BioReagent, for molecular biology, suitable for cell culture, suitable for plant cell culture, ≥99.5%
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L-Cysteine, from non-animal source, BioReagent, suitable for cell culture, ≥98%
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Urea, powder, BioReagent, for molecular biology, suitable for cell culture
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L-Cysteine, BioUltra, ≥98.5% (RT)
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Urea, BioUltra, for molecular biology, ≥99% (T)
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Boric acid, BioUltra, for molecular biology, ≥99.5% (T)
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Urea, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99%
SAFC
L-Cysteine
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Urea solution, 40 % (w/v) in H2O
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Urea-12C, 99.9 atom % 12C
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Andrographolide, 98%
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Boric acid-11B, ≥99 atom % 11B